The importance of analyzing gender politics within MSM communities is emerging in the field of sexuality and health studies. To advance this work, we examined gender expression (GE) heterogeneity among MSM in South Africa and its relationship to sexual health outcomes. Between October and December 2008, 480 gay and bisexual MSM (GBMSM) in South Africa were recruited using random driven-sampling (RDS). Participants were asked to complete an assessment of socio-demographic characteristics, feminine and masculine GE, and dating and sexual experiences. The present analysis was weighted to adjust for small vs. larger personal network size, yielding a total sample count of 496. Interaction effects associated with GE [continuous scores, sample mean score (M)=3.0, range=from masculine (1.0) to feminine (5.0)] by sexual identity (n=328 gay and n=129 bisexual) were examined using two-way analysis of variance (ANOVA) procedures. Participants were 67% young (18-24 years old) and were 31% HIV-positive (confirmed). GE was significantly associated with sexual identity (p<0.001), with gay MSM, on average, more feminine compared to bisexual MSM, [M=3.4, standard deviation (SD=0.9) vs. M=2.1 (SD=0.7), respectively]. Significant interaction in GE (p<0.05) were observed between GBMSM, and their socio-demographics (age, HIV status), dating experiences (male partners buying drinks, sharing regular partner with friends), and sexual experiences (sex with women, group sex, perceived GE of male sexual partner, and sexual pleasures in oral and anal sex).

BackgroundAssociation between the C677T polymorphism of the methylene tetrahydrofolate reductase (MTHFR) gene and congenital heart disease (CHD) is contentious.Methods and ResultsWe compared genotypes between CHD cases and controls and between mothers of CHD cases and controls. We placed our results in context by conducting meta-analyses of previously published studies. Among 5814 cases with primary genotype data and 10 056 controls, there was no evidence of association between MTHFR C677T genotype and CHD risk (odds ratio [OR], 0.96 [95% confidence interval, 0.87-1.07]). A random-effects meta-analysis of all studies (involving 7697 cases and 13 125 controls) suggested the presence of association (OR, 1.25 [95% confidence interval, 1.03-1.51]; P=0.022) but with substantial heterogeneity among contributing studies (I-2=64.4%) and evidence of publication bias. Meta-analysis of large studies only (defined by a variance of the log OR <0.05), which together contributed 83% of all cases, yielded no evidence of association (OR, 0.97 [95% confidence interval, 0.91-1.03]) without significant heterogeneity (I-2=0). Moreover, meta-analysis of 1781 mothers of CHD cases (829 of whom were genotyped in this study) and 19 861 controls revealed no evidence of association between maternal C677T genotype and risk of CHD in offspring (OR, 1.13 [95% confidence interval, 0.87-1.47]). There was no significant association between MTHFR genotype and CHD risk in large studies from regions with different levels of dietary folate.ConclusionsThe MTHFR C677T polymorphism, which directly influences plasma folate levels, is not associated with CHD risk. Publication biases appear to substantially contaminate the literature with regard to this genetic association.

BACKGROUND Autism spectrum disorder (ASD) represents a wide range of neurodevelopmental disorders characterized by impairments in social interaction, language, communication and range of interests. Autism is usually diagnosed in children 3-5 years of age using behavioral characteristics; thus, diagnosis shortly after birth would be beneficial for early initiation of treatment. AIM This retrospective study sought to identify newborns at risk for ASD utilizing bloodspot specimens in an immunoassay. MATERIALS & METHODS The present study utilized stored frozen specimens from ASD children already diagnosed at 15-36 months of age. The newborn specimens and controls were analyzed by immunoassay in a multiplex system that included 90 serum biomarkers and subjected to statisical analysis. RESULTS Three sets of five biomarkers associated with ASD were found that differed from control groups. The 15 candidate biomarkers were then discussed regarding their association with ASD. CONCLUSION This study determined that a statistically selected panel of 15 biomarkers successfully discriminated presumptive newborns at risk for ASD from those of nonaffected controls.

Background— Association between the C677T polymorphism of the methylene tetrahydrofolate reductase ( MTHFR ) gene and congenital heart disease (CHD) is contentious. Methods and Results— We compared genotypes between CHD cases and controls and between mothers of CHD cases and controls. We placed our results in context by conducting meta-analyses of previously published studies. Among 5814 cases with primary genotype data and 10 056 controls, there was no evidence of association between MTHFR C677T genotype and CHD risk (odds ratio [OR], 0.96 [95% confidence interval, 0.87–1.07]). A random-effects meta-analysis of all studies (involving 7697 cases and 13 125 controls) suggested the presence of association (OR, 1.25 [95% confidence interval, 1.03–1.51]; P =0.022) but with substantial heterogeneity among contributing studies (I 2 =64.4%) and evidence of publication bias. Meta-analysis of large studies only (defined by a variance of the log OR <0.05), which together contributed 83% of all cases, yielded no evidence of association (OR, 0.97 [95% confidence interval, 0.91–1.03]) without significant heterogeneity (I 2 =0). Moreover, meta-analysis of 1781 mothers of CHD cases (829 of whom were genotyped in this study) and 19 861 controls revealed no evidence of association between maternal C677T genotype and risk of CHD in offspring (OR, 1.13 [95% confidence interval, 0.87–1.47]). There was no significant association between MTHFR genotype and CHD risk in large studies from regions with different levels of dietary folate. Conclusions— The MTHFR C677T polymorphism, which directly influences plasma folate levels, is not associated with CHD risk. Publication biases appear to substantially contaminate the literature with regard to this genetic association.

Owing to technological advances during the last decade, there has been a paradigm shift in the way that we diagnose patients with inherited metabolic disease. The introduction of tandem mass spectrometry (MS/MS),7 a technology with sufficient analytical sensitivity to measure metabolic biomarkers in small blood spot samples, has allowed us to shift from what was initially a diagnosis made after or during acute metabolic decompensation into an era in which diagnosis is made immediately in the newborn period. In this environment, the diagnosis of certain metabolic diseases frequently precedes the onset of symptoms, giving physicians an opportunity to prevent many of the catastrophic events that used to occur. To this point, disorders such as medium-chain acyl-CoA dehydrogenase (MCAD) deficiency now rarely initially present as the acute-onset liver failure that was previously seen in unscreened and consequently undiagnosed patients. Clinical outcomes for MCAD deficiency are now excellent, and data on some of the other screened disorders are starting to show the benefit of early diagnosis. Diagnostic technology continues to increase in its capability to provide early diagnosis of increasing numbers of these metabolic diseases as more biomarkers become identified. In this Q&A, 5 leading thinkers in the field provide their insights into where we presently stand in this area and where we might be headed in the future. Has there been a positive impact on patient care as a result of early diagnosis and implementation of the American College of Medical Genetics (ACMG) recommended-screening disorders? Piero Rinaldo: The implementation of the ACMG uniform panel has had a positive impact at multiple levels. The consistency of the panel has virtually eliminated the discrepancies between neighboring states, often a cause of heartbreaking events where parents have experienced significant morbidity and mortality of their child that would not have happened if their child …

Biotinidase deficiency is an autosomal recessive syndrome caused by defects in the biotinidase gene, the product of which affects biotin metabolism. Newborn screening (NBS) for biotinidase deficiency can identify affected infants prior to onset of symptoms; biotin supplementation can resolve or prevent the clinical features. In NBS, dry blood spots (DBS) are usually tested for biotinidase enzyme activity by colorimetric analysis. By taking advantage of the multiplexing capabilities of the Luminex platform, we have developed a microsphere-based array genotyping method for the simultaneous detection of six disease causing mutations in the biotinidase gene, thereby permitting a second tier of molecular analysis. Genomic DNA was extracted from 3.2 mm DBS. Biotinidase gene sequences, containing the mutations of interest, were amplified by multiplexed polymerase chain reaction, followed by multiplexed allele-specific primer extension using universally tagged genotyping primers. The products were then hybridized to anti-tag carrying xTAG microspheres and detected on the Luminex platform. Genotypes were verified by sequencing. Genotyping results of 22 known biotinidase deficient samples by our xTAG biotinidase assay was in concordance with the results obtained from DNA sequencing, for all 6 mutations used in our panel. These results indicate that genotyping by an xTAG microsphere-based array is accurate, flexible, and can be adapted for high-throughput. Since NBS for biotinidase deficiency is by enzymatic assay, less than optimal quality of the DBS itself can compromise enzyme activity, while the DNA from these samples mostly remains unaffected. This assay warrants evaluation as a viable complement to the biotinidase semi-quantitative colorimetric assay.

Purpose: To achieve clinical validation of cutoff values for newborn screening by tandem mass spectrometry through a worldwide collaborative effort. Methods: Cumulative percentiles of amino acids and acylcarnitines in dried blood spots of approximately 25–30 million normal newborns and 10,742 deidentified true positive cases are compared to assign clinical significance, which is achieved when the median of a disorder range is, and usually markedly outside, either the 99th or the 1st percentile of the normal population. The cutoff target ranges of analytes and ratios are then defined as the interval between selected percentiles of the two populations. When overlaps occur, adjustments are made to maximize sensitivity and specificity taking all available factors into consideration. Results: As of December 1, 2010, 130 sites in 45 countries have uploaded a total of 25,114 percentile data points, 565,232 analyte results of true positive cases with 64 conditions, and 5,341 cutoff values. The average rate of submission of true positive cases between December 1, 2008, and December 1, 2010, was 5.1 cases/day. This cumulative evidence generated 91 high and 23 low cutoff target ranges. The overall proportion of cutoff values within the respective target range was 42% (2,269/5,341). Conclusion: An unprecedented level of cooperation and collaboration has allowed the objective definition of cutoff target ranges for 114 markers to be applied to newborn screening of rare metabolic disorders.

To the Editor: Testing for severe combined immunodeficiency (SCID) has recently become of interest to the newborn-screening community, because the detection of this condition in early infancy is crucial for effective treatment. Several newborn-screening programs have implemented screening for this condition. The current screening method for SCID is by the T-cell receptor excision circles assay (1), which detects the presence or absence of an excised segment of DNA; however, molecular techniques are not routinely adopted in many newborn-screening laboratories, where an immunoassay would be preferred. As recently reported (2), we developed an immunoassay that quantifies CD3, a component of the T-cell receptor complex (3), to provide an alternative or complementary method to T-cell receptor excision circles testing. Low or absent T cells in the peripheral blood is a common characteristic of T-cell immunodeficiencies and of all but one form of SCID. We have previously described an immunoassay using the Luminex microsphere technology that displays excellent potential for the detection of CD3 from dry blood spot samples (2). We report the validation of an improved …

1. Chan K, Puck JM. Development of populationbased newborn screening for severe combined immunodeficiency. J Allergy Clin Immunol 2005; 115:391–8. 2. Janik DK, Lindau-Shepard B, Comeau AM, Pass KA. A multiplex immunoassay using the Guthrie specimen to detect T-cell deficiencies including severe combined immunodeficiency disease. Clin Chem 2010;56:1460–5. 3. Dave VP. Hierarchical role of CD3 chains in thymocyte development. Immunol Rev 2009; 232:22–33. 4. Nørgaard-Pedersen B, Hougaard DM. Storage policies and use of the Danish Newborn Screening Biobank. J Inherit Metab Dis 2007;30: 530–6. 5. Larsson A, Bålöw RM, Lindahl TL, Forsberg PO. Chicken antibodies: taking advantage of evolution—a review. Poult Sci 1993;72:1807– 12.

Primary congenital hypothyroidism (CH) is a common and preventable cause of intellectual disability. The incidence rate of CH has been reported to be increasing in the United States, but the factors behind the observed rate increase are not known. We summarize here the data presented at a workshop on CH, at which factors potentially related to the CH-incidence-rate increase (namely, race, ethnicity, sex, and birth outcomes) were evaluated. Data sources for the analyses included a national data set of newborn-screening results and state-specific data from newborn-screening programs in California, Massachusetts, New York, and Texas. The incidence rate of CH increased in the United States by 3% per year; however, an increase did not occur in all states, at a constant rate, or even at the same rate. Analysis of US data (1991–2000) showed a CH-incidence-rate increase only among white newborns. More recently, in California (2000–2007), the rate was constant in non-Hispanic newborns, but it increased among Hispanic newborns. In the national data, the CH-incidence rate increased similarly among boys and girls, whereas in Texas (1992–2006), the rate among boys increased significantly more than among girls and varied according to race and ethnicity. In Massachusetts (1995–2007), low birth weight newborns or newborns who had a delayed rise in thyrotropin concentration accounted for the majority of the recent rate increase. Race, ethnicity, sex, and pregnancy outcomes have affected the observed increasing incidence rate of CH, although there have been some inconsistencies and regional differences. The association with preterm birth or low birth weight could reflect the misclassification of some cases of transient hypothyroxinemia as true CH. Future studies of risk factors should focus on correct initial identification and reporting of demographic characteristics and pregnancy outcomes for cases of CH. In addition, long-term follow-up data of presumed cases of CH should be ascertained to differentiate true cases of CH from cases of transient hypothyroidism.

Patients with rare and complex diseases such as congenital adrenal hyperplasia (CAH) often receive fragmented and inadequate care unless efforts are coordinated among providers. Translating the concepts of the medical home and comprehensive health care for individuals with CAH offers many benefits for the affected individuals and their families. This manuscript represents the recommendations of a 1.5 day meeting held in September 2009 to discuss the ideal goals for comprehensive care centers for newborns, infants, children, adolescents, and adults with CAH. Participants included pediatric endocrinologists, internal medicine and reproductive endocrinologists, pediatric urologists, pediatric surgeons, psychologists, and pediatric endocrine nurse educators. One unique aspect of this meeting was the active participation of individuals personally affected by CAH as patients or parents of patients. Representatives of Health Research and Services Administration (HRSA), New York-Mid-Atlantic Consortium for Genetics and Newborn Screening Services (NYMAC), and National Newborn Screening and Genetics Resource Center (NNSGRC) also participated. Thus, this document should serve as a "roadmap" for the development phases of comprehensive care centers (CCC) for individuals and families affected by CAH.

BACKGROUND:Severe combined immunodeficiency (SCID) fulfills many of the requirements for addition to a newborn screening panel. Two newborn screening SCID pilot studies are now underway using the T-cell receptor excision circle (TREC) assay, a molecular technique. Here we describe an immunoassay with CD3 as a marker for T cells and CD45 as a marker for total leukocytes that can be used with the Guthrie specimen. METHODS:The multiplexing capabilities of the Luminex platform were used. Antibody pairs were used to capture and detect CD3 and CD45 from a single 3-mm punch of the Guthrie specimen. The assay for each biomarker was developed separately in identical buffers and then combined to create a multiplex assay. RESULTS:Using calibrators made from known amounts of leukocytes, a detection limit of 0.25 x 10(6) cells/mL for CD3 and 0.125 x 10(6) cells/mL for CD45 was obtained. Affinity tests showed no cross-reactivity between the antibodies to CD3 and CD45. The multiplex assay was validated against 8 coded specimens of known clinical status and linked to results from the TREC assay that had identified them. All were correctly identified by the CD345 assay. CONCLUSIONS:The performance parameters of the CD345 assay met the performance characteristics generally accepted for immunoassays. Our assay classifications of positive specimens concur with previous TREC results. This CD345 assay warrants evaluation as a viable alternative or complement to the TREC assay as a primary screening tool for detecting T-cell immunodeficiencies, including SCID, in Guthrie specimens.

BACKGROUND:Real-time quantitative PCR (qPCR) targeting a specific marker of functional T cells, the T-cell-receptor excision circle (TREC), detects the absence of functional T cells and has a demonstrated clinical validity for detecting severe combined immunodeficiency (SCID) in infants. There is need for a qPCR TREC assay with an internal control to monitor DNA quality and the relative cellular content of the particular dried blood spot punch sampled in each reaction. The utility of the qPCR TREC assay would also be far improved if more tests could be performed on the same newborn screening sample.METHODS:We approached the multiplexing of qPCR for TREC by attenuating the reaction for the reference gene, with focus on maintaining tight quality assurance for reproducible slopes and for prevention of sample-to-sample cross contamination. Statewide newborn screening for SCID using the multiplexed assay was implemented, and quality-assurance data were recorded.RESULTS:The multiplex qPCR TREC assay showed nearly 100% amplification efficiency for each of the TREC and reference sequences, clinical validity for multiple forms of SCID, and an analytic limit of detection consistent with prevention of contamination. The eluate and residual ghost from a 3.2-mm dried blood spot could be used as source material for multiplexed immunoassays and multiplexed DNA tests (Multiplex Plus), with no disruption to the multiplex TREC qPCR.CONCLUSIONS:Population-based SCID newborn screening programs should consider multiplexing for quality assurance purposes. Potential benefits of using Multiplex Plus include the ability to perform multianalyte profiling.

Congenital hypothyroidism and congenital adrenal hyperplasia are included in many newborn screening (NBS) panels worldwide and in all state-sponsored programs in the United States. Both conditions meet the fundamental prerequisites for NBS: high incidence in the population; biomarkers in the dried blood specimen that are easily detected; and, effective therapies to lessen, if not prevent, the sequelae of late or no treatment. In this review, the history of NBS is discussed for these 2 conditions. The technologies and protocols used in their detection, and related subjects such as genetics, and treatment and outcomes, are also discussed.

The use of molecular genetic tests for heritable conditions is expected to increase in medical settings, where genetic knowledge is often limited. As part of a project to improve the clarity of genetic test result reports to minimize misunderstandings that could compromise patient care, we sought input about format and content from practicing primary care clinicians. In facilitated workgroup discussions, clinicians from pediatric, obstetrics-gynecology, and family practice provided their perspectives about molecular genetic testing with a focus on the laboratory reporting of test results. Common principles for enhancing the readability and comprehension of test result reports were derived from these discussions. These principles address the presentation of patient- and test-specific information, the test result interpretation, and guidance for future steps. Model test result reports for DNA-based cystic fibrosis testing are presented that were developed based on workgroup discussions, previous studies, and professional guidelines. The format of these model test reports, which are applicable to a variety of molecular genetic tests, should be useful for communicating essential information from the laboratory to health care professionals.

Fetal Alcohol Spectrum Disorder (FASD) is one of the most common causes of acquired mental retardation in the United States and worldwide. The fetal brain is highly susceptible to the teratogenic effects of alcohol from maternal consumption during pregnancy resulting in newborns with mental deficits and congenital malformations. FAS diagnosis is difficult to diagnose in newborns where distinct anatomical defects are not apparent from mothers of moderate to light alcohol use. Hence, medical diagnoses are often not ascertained until mid-childhood after irreparable brain damage has already occurred. Such infants will have been deprived of available socioclinical interventions, trainings, measures, and future treatments that may someday be implemented soon after birth. Presently, there are no FASD newborn biomarker screening programs in place despite cost benefit analyses revealing an annual societal cost of $1.3 million per FASD incident case. Since newborn biomarkers have been reported in the biomedical literature, can we afford not to implement newborn screening for FASD?

Patients identified in newborn screening programs can be among the most vulnerable during a disaster due to their need to have prompt diagnosis and medical management. Recent disasters have challenged the ability of newborn screening programs to maintain the needed continuity during emergency situations. This has significant implications for the newborn screening laboratories, the diagnostic confirmation providers, and the patients who either require diagnosis or maintenance of their therapeutic interventions. In 2007, the National Coordinating Center (NCC) for the Regional Genetics and Newborn Screening Collaboratives (RCs) sponsored a meeting involving representatives of the Regional Genetics and Newborn Screening Collaborative Groups, state newborn screening programs, providers of diagnosis and confirmation services, manufacturers of equipment, medical foods, and other treatments used in patients identified in newborn screening programs, and individuals from agencies involved in disaster response including the National Disaster Medical Service, the Centers for Disease Control and Prevention, the Emergency Management Assistance Compact, the Federal Emergency Management Agency, and others. In addition to developing contingency plans for newborn screening, we have considered other uses of genetics as it is used in DNA-based kinship identification of mass casualties. The meeting resulted in the description of a wide range of issues facing newborn screening programs, provider groups, and patients for which emergency preparedness development is needed in order that appropriate response is enabled.

BACKGROUND Since its beginnings, newborn screening for cystic fibrosis (CF) using an assay for immunoreactive trypsinogen (IRT) has been plagued by a high rate of false-positive results (screen positive, diagnosis negative), despite attempts to reduce this rate by use of altered cutoffs and second-tier DNA testing. IRT exists as 2 isoforms: IRT1 and IRT2, with IRT2 being more closely aligned with pancreatic disease, including CF. Assay standardization between programs is a continuing problem because the IRT assays currently in use variously recognize either 1 or both isoforms. Here we report the development of a multiplexed assay for both forms of IRT simultaneously. METHODS Using 2 different Luminex bead sets, we developed assays for each IRT isoform separately and then combined them. Using the sum of IRT1 and IRT2 values (IRT1+IRT2), we compared the results with a CF kit currently in use. RESULTS In a sample set consisting of 16 cases confirmed positive for CF, we established a cutoff at >97 microg/L total IRT. Seven of 8 carriers with 1 CF mutation screen-positive by the standard method were also screen-positive by IRT1+IRT2. Of 32 cases screen-positive by standard IRT, 11 were screen-negative by IRT1+IRT2. None of these 11 cases had CF mutations identified by the screening program. CONCLUSIONS These data indicate that the multiplex method with specificity for 2 isoforms of IRT has performance comparable to that of a standard IRT method and the advantage of improved standardization by detection of the 2 isoforms.